The isoelectric point (hereinafter "pI") is an important parameter which is useful in determining the acid-base properties of amphoteric molecules such as proteins and polypeptides. The pI is generally defined as the pH value at which the net overall charge on the molecule is zero. A knowledge of the pI for a particular amphoteric substance, therefore, aids the investigator in selecting between the ever increasing number of protein isolation techniques to obtain and purify an amphoteric molecule of interest. Some of the better known isolation techniques include isoelectric focusing, chromatofocusing, isoelectric precipitation, disc electrophoresis, isotachophoresis, ion-exchange chromatography and ammonium sulfate fractionation. In particular, with the advent of isoelectric focusing and chromatofocusing, pI data for proteins and polypeptides have been accumulated and enlarged such that more than 1,000 of protein pI values have been determined over the past two decades [Righetti et al., J. Chromatogr. 127:1-28 (1976); Righetti et al., J. Chromatogr. 220:116-194 (1981); Malamud et al., Anal. Biochem. 86:620-647 (1978)].
Despite the need for useful pI data to aid in the use of the above-identified protein isolation techniques, the present methods for generally determining pI such as isoelectric focusing or chromatography present several drawbacks. One is the excessively long processing time required to utilize these methods. Another is the effect of the processing time on the accuracy and precision of the method itself; for example, when isoelectric focusing is used, the isoelectric point (as measured by the pH at which a protein zone is found) varies as a function of the electrofocusing time during a transient phase of indeterminant length [Horuk et al., FEBLETT. 155:213-217 (1983); An Der Lan et al., Arch. Biochem. Biophys. 200:206-215 (1980)]. This period of electrofocusing time must be measured for each individual protein under test before any accurate pI value can be determined. A third drawback is that pI determinations obtained by presently known methods are usually limited by a strict requirement that they be performed at 4.degree. C.; the 4.degree. C. environment is maintained in order to lessen the risk of protein denaturation resulting from either the long focusing times or the high electric field involved. The difficulty arises because the pI value of a protein depends upon the dissociation constants (pK) of the ionizable amino acids present and is, therefore, temperature dependent; accordingly, a pI value obtained at 4.degree. C. is not necessarily accurate or indicative of the true pI value for that protein at any other temperature. A fourth deficiency is that unless a gel containing immobilized carrier ampholytes is used as part of the methodology, the presence of carrier ampholytes in the medium can interact with the proteins under test and cause the formation of complexes and artifacts [Righetti et al., Biochem. Biophys. Acta. 532:137-146 (1978); Gianazza et al., in Electrophoresis (Radola, B. J., Ed.) Vol. 79,1980, pp 129-140; Shinjo et al., FEBS Letters 105:353-356 (1979)].
Alternative methods for determining the isoelectric point for amphoteric molecules have been investigated. Of these, the report of Lampson and Tytell [Anal. Biochem. 11:374-377 (1965)] that the pI of a protein could be determined by CM-Sephadex column chromatography using buffers with increasing pH as an eluent is the most pertinent. The Lampson and Tytell technique has several inherent deficiencies: the method requires multiple chromatographic procedures and is very time consuming; by the nature of the ion-exchange material used by the investigators, the method is limited for use to only those proteins having an isoelectric point above pH 6.0; in addition, deviations of 0.4-0.6 pH units from the literature values for each of the proteins tested was empirically obtained using this method. For these reasons, there remains an apparent and longstanding need for methods to determine the isoelectric point of proteins, polypeptides and other molecules which is simple to perform, rapid in processing time, provides accuracy and reproductibility with a minimum deviation of results and is both versatile and available to amphoteric molecules in general without limitation.